Since their discovery as key mediators of cytokine signaling, considerable progress has been made in de®ning the structure-function relationships of Signal Transducers and Activators of Transcription (STATs). In addition to their central roles in normal cell signaling, recent studies have demonstrated that diverse oncoproteins can activate speci®c STATs (particularly Stat3 and Stat5) and that constitutively-activated STAT signaling directly contributes to oncogenesis. Furthermore, extensive surveys of primary tumors and cell lines derived from tumors indicate that inappropriate activation of speci®c STATs occurs with surprisingly high frequency in a wide variety of human cancers. Together, these ®ndings provide compelling evidence that aberrant STAT activation associated with oncogenesis is not merely adventitious but instead contributes to the process of malignant transformation. These studies are beginning to reveal the molecular mechanisms leading to STAT activation in the context of oncogenesis, and candidate genes regulated by STATs that may contribute to oncogenesis are being identi®ed. Recent studies suggest that activated STAT signaling participates in oncogenesis by stimulating cell proliferation and preventing apoptosis. This review presents the evidence for critical roles of STATs in oncogenesis and discusses the potential for development of novel cancer therapies based on mechanistic understanding of STAT signaling.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that were first identified as mediators of cellular responses to interferons (reviewed in references 12, 16 and 35). Signaling induced by the interaction of interferons and other cytokines with their cognate receptors is initiated by a cascade of events, including receptor aggregation and activation of Janus protein tyrosine kinases (JAKs) associated with the receptors. Subsequently, STAT proteins are recruited to the receptor-JAK complexes and activated by tyrosine phosphorylation, which promotes the formation of homodimers or heterodimers of STAT family members. Activated STATs, in turn, translocate to the nucleus and bind to specific DNA response elements that regulate gene expression. There are at least seven genes in the mammalian genome known to encode different STAT family members, which are activated in various combinations in response to stimulation by numerous cytokines (12,16,35).It has become evident that, in addition to cytokines, mitogenic growth factors, such as platelet-derived growth factor and epidermal growth factor, also induce STAT signaling, particularly Stat1, Stat3, and Stat5 (21,35). An emerging concept is that normal signaling by STAT proteins is involved in control of diverse biological processes regulated by cytokines and growth factors, including cell differentiation, proliferation, development, and apoptosis (2,

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological eects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our ®ndings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

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Signal transducers and activators of transcription (STATs) were originally discovered as latent cytoplasmic transcription factors that mediate cellular responses to diverse cytokines and growth factors (for reviews, see references 17, 18, and 55). STATs are activated by tyrosine phosphorylation, dimerize, and subsequently translocate to the nucleus, where they regulate the transcription of genes by binding to specific DNA response elements. Studies have implicated normal STAT signaling in controlling fundamental biological processes, including cell differentiation, proliferation, apoptosis, and development (7,15,26,33,60,78). Multiple signaling pathways are simultaneously induced in response to cytokine or growth factor stimulation, consistent with complex regulation by signal cross talk. For example, maximum transcriptional activity of certain STATs requires serine phosphorylation mediated by serine/threonine kinases of other signaling pathways (3,19,51,68). The kinases that mediate STAT serine phosphorylation are not fully defined, although evidence implicates multiple serine kinase signals, including mitogen-activated protein kinases (MAPKs)/extracellular signal-regulated kinases (ERKs) (19), an H7-sensitive serine kinase (5), and a MAPK kinase (MKK)-dependent, ERK-independent serine kinase (11).MAPKs represent a family of serine/threonine protein kinases comprising ERK1/ERK2 (ERKs), p38/HOG1 (p38), and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) (reviewed in references 24, 43, and 59). Ras and Raslike small G proteins are key regulators in the signaling pathways leading to MAPK activation. For the Ras-ERK branch, sequential protein phosphorylations are mediated by the serine/threonine kinase Raf-1 and the dual-specificity MKKs, which in turn phosphorylate and activate ERKs (24,48,49,72). For the JNK and p38 pathways, the Rac1/Cdc42 subfamily of small G proteins is a key mediator, together with Ras (for reviews, see references 24, 43, and 59). Several serine/threonine protein kinases that are members of the mixed-lineage kinases (MLK), such as dual leucine-zipper bearing kinase (DLK), have been identified as upstream activators of MKKs (23,24,38). Activation of JNK is largely induced by MKK4 and MKK7, while MKK3 and MKK6 preferentially activate p38 (22,24,62,75). Activated MAPKs ultimately phosphorylate transcription factors in the nucleus that are responsible for the regulation of immediate-early genes, such as c-fos, whose functional roles include control of cell proliferation (35,37,71).Emerging evidence strongly implicates abnormal activation of STAT signaling in oncogenic transformation. Our laboratory and others have previously reported constitutively active

While the activated viral Src oncoprotein, v-Src, induces uncontrolled cell growth, the mechanisms underlying cell cycle deregulation by v-Src have not been fully de®ned. Previous studies demonstrated that v-Src induces constitutively active STAT3 signaling that is required for cell transformation and recent data have implicated STAT3 in the transcriptional control of critical cell cycle regulators. Here we show in mouse ®broblasts stably transformed by v-Src that mRNA and protein levels of p21 (WAF1/CIP1), cyclin D1, and cyclin E are elevated. Using reporter constructs in transient-transfection assays, the cyclin D1 and p21 promoters were both found to be transcriptionaly induced by v-Src in a STAT3-dependent manner. The kinase activities of cyclin D/ CDK4, 6 and cyclin E/CDK2 complexes were only slightly elevated, consistent with the ®ndings that coordinate increases in p21, cyclin D1 and cyclin E resulted in an increase in cyclin/CDK/p21 complexes. Similar results were obtained in NIH3T3 and BALB/c 3T3 cells stably transformed by v-Src, indicating that these regulatory events associated with STAT3 signaling represent common mechanisms independent of cell line or clonal variation. These ®ndings suggest that STAT3 has an essential role in the regulation of critical cell cycle components in v-Src transformed mouse ®broblasts.